REGULATION OF MINERALIZATION IN SKELETAL TISSUES

生物矿化在骨骼和牙齿发育过程中起着至关重要的作用，并使骨骼组织在成年期发挥其正常功能。尽管其重要性显而易见，但人们对其监管却知之甚少。组织中矿物质形成的调节对其正常功能至关重要。例如，过量的矿物质沉积伴随着动脉粥样硬化和骨关节炎。在后者中，关节表面的晶体形成可能在炎症的发生和关节破坏的进展中起主要作用。另一方面，骨质疏松症中所见的矿化损失会导致脆弱的骨骼和骨折。基质囊泡是一种小的膜封闭颗粒，在许多组织中起着启动矿化的关键作用，包括颅面骨、长骨、软骨和牙本质。虽然已经确定这些颗粒是从质膜中释放出来的，但目前尚不清楚基质囊泡的释放是如何被调节的，以及在它们释放到细胞外基质后，囊泡是如何引发矿物质形成的。在我们的初步研究中，首次表明只有矿化的细胞释放膜联蛋白II和富含v，含有Ca2+/Pi复合物（核核）的基质囊泡能够启动矿化，而非矿化细胞释放的囊泡不会矿化。此外，我们提供的证据表明，膜联蛋白II和V在基质囊泡中形成Ca2+通道，允许Ca2+流入这些颗粒和第一个腔内晶体的形成和生长。这些膜联蛋白II和富含V的基质囊泡的释放伴随着膜联蛋白II和V的表达和细胞质Ca2+浓度的增加，[Ca2+]i。我们的假设是(i)细胞质Ca2+浓度和膜联蛋白II和V表达的增加是释放矿化能力强的基质囊泡所必需的，（II）膜联蛋白II和V与核核合作，使Ca2+流入囊泡，形成第一个矿相和矿化的开始。该项目将通过使用多种实验策略直接测试这些假设，包括细胞培养，Ca2+通道研究，定点诱变和突变膜联蛋白分子的表达。目前的建议将提供新的见解，一些最有趣的和重要的特征，调节矿化在体内。这一信息将是至关重要的，以防止不受控制的矿化在若干组织病理条件下。

Biomineralization plays a crucial role during skeletal and tooth development and allows skeletal tissues to exert their proper functions during adulthood. Despite its obvious importance little is known about its regulation. Regulation of mineral formation in tissues is critical for their proper function. For example, excessive mineral deposition accompanies atheriosclerosis and osteoarthritis. In the latter, crystal formation in the articular surface area may play a major role in the onset of inflammation and the progression of joint destruction. On the other hand, loss of mineralization as seen in osteoporosis leads to fragile bones and bone fractures. Matrix vesicles, small membrane-enclosed particles, have the critical role of initiating mineralization in many tissues, including craniofacial bones, long bones, cartilage and dentin. While it is well established that these particles are released from the plasma membrane, it is unclear how the release of matrix vesicles is regulated and how, following their release into the extracellular matrix, vesicles initiate mineral formation. In our Preliminary Studies it is shown for the first time that only cells undergoing mineralization release annexins II and V-rich, Ca2+/Pi complexes (nucleational core)-containing matrix vesicles which are able to initiate mineralization, while nonmineralizing cells release vesicles which do not mineralize. In addition, we provide evidence that annexins II and V form Ca2+ channels in matrix vesicles, allowing Ca2+ influx into these particles and the formation and growth of the first intralumenal crystals. Release of these annexins II and V-rich matrix vesicles is accompanied by increases in annexins II and V expression and cytosolic Ca2+ concentration, [Ca2+]i. Our hypotheses are that (i) increases in both cytosolic Ca2+ concentration and annexins II and V expression are required for the release of mineralization-competent matrix vesicles, and that (ii) annexins II and V cooperate with the nucleational core to enable Ca2+ influx into the vesicles, formation of the first mineral phase and initiation of mineralization. This project will test these hypotheses directly by the use of diverse experimental strategies, including cell culture, Ca2+ channel studies, site-directed mutagenesis and expression of mutated annexin molecules. The present proposal will provide new insights into some of the most intriguing and important features which regulate mineralization in vivo. This information will be of critical importance for the prevention of uncontrolled mineralization in several tissues during pathological conditions.